The selective, non-covalent modification of recombinant proteins with spectroscopic or microscopic probes or (bio)chemically functional units is a central challenge for proteome-analysis and biotechnological uses. In principle, such modifications can be realized with local specificity by means of so-called affinity-tags, i.e. short peptide sequences that are introduced into a protein by genetic engineering. These affinity-tags are specifically recognized by (bio)chemical recognition units. While for purification several affinity-tags were used very successfully, their use for the attachment of spectroscopic or microscopic probes and other biochemical functional units in solution and to surfaces is often critical, since the complexes exhibit an insufficient stability.
Since the middle of the seventies, the chelator iminodiacetic acid (IDA) has already been used in combination with several metal ions for purifying of proteins (Porath J, Carlsson J, Olsson I, Belfrage G. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258(5536):598-9.).
In the middle of the eighties, nitrilotriacetic acid (NTA) was described as a chelator for purifying of proteins (EP 0 253 303 B1, U.S. Pat. No. 4,877,830). Furthermore, the (cumulated) histidine-tag was described for the generic purification of recombinant proteins (Hochuli E, Dobel H, Schacher A. 1987. New metal chelate adsorbent selective for proteins and peptides containing neighboring histidine residues. J Chromatogr 411:177-84; see EP 0 282 042 B1, U.S. Pat. No. 5,284,933). Today, the histidine-tag with is by far the most commonly used affinity-tag, and most diverse matrices and detection techniques that rely on the interaction of oligohistidin with chelator-bound metal ions are described (Ueda E K, Gout P W, Morganti L. 2003. Current and prospective applications of metal ion-protein binding. J Chromatogr A 988(1):1-23.). Nevertheless, the binding affinity of individual Ni-NTA-oligohistidine-interactions is too low in order to ensure a stable and stoichiometrically defined binding. A partially stable binding can be achieved through a high density of NTA-groups in an affinity matrix or a planar surface (see e.g. Dorn I. T., Pawlitschko K., Pettinger S. C., Tampe R. 1998. Orientation and two-dimensional organization of proteins at chelator lipid interfaces. Biol Chem. 379(8-9):1151-9.; Frenzel A, Bergemann C, Kohl G, Reinard T. 2003. Novel purification system for 6xHis-tagged proteins by magnetic affinity separation. J Chromatogr B Analyt Technol Biomed Life Sci. 793(2):325-9.; Paborsky L R, Dunn K E, Gibbs C S, Dougherty J P. 1996. A nickel chelate microtiter plate assay for six histidine-containing proteins. Anal Biochem. 234(1):60-5.; Lauer S A, Nolan J P. 2002. Development and characterization of Ni-NTA-bearing microspheres. Cytometry. 48(3):136-45.) For many uses, in particular for the manipulation of proteins in solution or in vivo, such as in living cells, nevertheless, a high binding stability at a molecular level is required.
First, in order to improve the binding between affinity-tag and metal-chelator, it is one possibility to manipulate the affinity-tag. Thus, on the one hand, extended histidine-tags are described, such as by Guiget et al., which use 10 histidine-residues instead of the common 6 (Guignet E G, Hovius R, Vogel H. 2004. Reversible site-selective labeling of membrane proteins in live cells. Nat Biotechnol. 22(4):440-4.). Nevertheless, in this maimer neither stoichiometrically defined nor stable complexes can be achieved. On the other hand, proteins and protein complexes are described that carry two histidine-tags and therefore bind more stably to the surfaces of Ni-NTA-chips (Nieba L, Nieba-Axmann S E, Persson A, Hamalainen M, Edebratt F, Hansson A, Lidholm J, Magnusson K, Karlsson A F, Plückthun A. 1997. BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem. 252(2):217-28.). Nevertheless, not all proteins can be provided with more than one affinity-tag at their termini without too much affecting their biological functions.
The other possibility in order to increase the binding consists of improving the chelator group itself. Thus, Ebright and Ebright in WO 03/091689 (and in Kapanidis A N, Ebright Y W, Ebright R H. 2001. Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (Ni(2+):nitrilotriacetic Acid (n)-fluorochrome conjugates. J Am Chem Soc. 123(48):12123-5.) describe bivalent chelator complexes for in situ labelling of proteins with a detectable group, such as, for example, a fluorophor. These complexes show an improved affinity for His-tags, compared to the monovalent complexes. The two chelator-groups (NTA), nevertheless, are directly bound at the detectable group, making them not universally accessible for synthesis. Rather, the possibility for their production will always depend from the synthetic compatibility of the selected detectable group.
Other bifunctional carboxymethyl-substituted chelators are described by Kline et al. (Kline S J, Betebenner D A, Johnson D K. 1991. Carboxymethyl-substituted bifunctional chelators: preparation of aryl isothiocyanate derivatives of 3-(carboxymethyl)-3-azapentanedioic acid, 3,12-to(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid, and 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid for use as protein labels. Bioconjug Chem. 2(1):26-31.). These chelators can covalenty bind the target protein, and were developed for a labelling with metal-complexes with catalytic activity.
Another strategy for a stable and selective labelling or modification of target proteins is described by Griffin et al. (Griffin B A, Adams S R, Tsien R Y. 1998. Specific covalent labeling of recombinant protein molecules inside live cells. Science. 281(5374):269-72.). Bi-arsenic-complexes are disclosed that specifically recognize a particular motif in recombinant proteins containing four cysteines (Cys-Cys-Xaa-Xaa-Cys-Cys). The bi-arsenic-complexes can contain detectable groups such as fluorophores, and thus modify or label the target proteins. This technology is also described by Tsien et al. in U.S. Pat. No. 6,008,378, and by Ebright and Ebright in WO 03/107010. The use of these bi-arsenic-complexes, nevertheless, is limited by the laborious synthesis of the respective bi-arsenic-derivatives as well as by the fact that only particular fluorophores and chromophores are possible. In addition, the cysteine-rich affinity-tags or motifs as required are disadvantageous for many uses due to the high reactivity of free thiol groups.
It is therefore the object of the present invention to provide multivalent chelators for binding to affinity-tags that are known and widely used in the state of the art, such as the oligo-histidine-tag, that exhibit a stoichiometric, stable interaction with the affinity-tag and interact switchable and reversible with the target molecule. By this, target molecules can be generically modified with a multitude of probes and other biochemically functional units in a selective and position-specific manner. Furthermore, the multivalent chelators shall be synthetically accessible in such a way that a controlled and universal conjugation with a multitude of probes and other biochemically functional units is possible.
According to the invention, this object is solved by providing of compounds of the general formulaXm-G-CLn                 wherein        G is a scaffold-structure,        X is a coupling group for a probe or functional unit F,        CL is a chelator-group with at least a metal-coordinative centre,        m is an integer and at least 1, and        n is an integer and at least 2,        and tautomers, isomers, anhydrides, acids and salts thereof.        
In a preferred embodiment, the scaffold-structures G comprise a saturated hydrocarbon chain with 2 to 25 carbon-atoms, preferred 2 to 20 and further preferred 5 to 16 carbon-atoms. Furthermore, the scaffold-structures comprise amide-, ester- and/or ether bonds.
The scaffold-structure can be linear, branched or closed. A preferred closed scaffold-structure is represented by a cyclam ring structure. Preferred scaffold-structures comprise amino acid-elements.
In a preferred embodiment, at least one metal ion is bound to each of the chelator-groups of the compounds according to the invention. Thereby, the metal ion is selected from the group consisting of Ni2+, Co2+, Cu2+, Zn2+, Fe2+, Fe3+, and all lanthanidions.
The chelator-group is preferably selected from the group consisting of nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), all variants of porphyrine systems, salicylic acid derivatives, 1,2-diaminoethyldiacetic acid, diaminoethyl triacetic acid, hydroxy ethylimino diacetic acid, and salts or combinations thereof, and other chelator-groups known to the person of skill.
In a preferred embodiment, the reactive groups of the chelator-group carry protective groups. For example, carboxyl-groups such as those of NTA can be protected by the OtBu-group. All common protective groups known to the person of skill can be used.
In a further embodiment a spacer group A is located between the chelator-groups and the scaffold-structure. The spacer group A can be linear or branched. The usual spacer groups that are known to the person of skill can be used. Preferred spacer groups comprise poly(ethylene glycol), oligo(ethylene glycol), peptides, (CH2)n, wherein n is from 1 to 8, oligoproline.
It is preferred that the chelator-groups are bound to the scaffold-structure through amide, ester or ether bonds.
According to the invention, it is preferred that the scaffold-structure G itself is not a probe or another detectable group. Preferably, G is a structure at which the chelator-groups and probes or functional units are attached.
Preferably, the compounds according to the invention are selected from
and from the tautomers, isomers, anhydrides, acids and salts thereof.
Preferred coupling groups X are selected from the group consisting of NHR, wherein R is H, an alkyl or aryl residue, COOH, (CH2)n—COOH, wherein n is an integer, SH, maleinimide, iodoacetamide, isothiocyanate or cyanate.
In a preferred embodiment, a probe or functional unit F is bound at the coupling group X of the compound according to the invention. The probe or functional unit F is preferably selected from the group consisting of fluorophores, FRET-fluorophores, fluorescence quenchers, phosphorescent compounds, luminescent compounds, absorbing compounds, polymers, PEG, oligosaccharides, oligonucleotides, PNA, biotin, haptenes, peptides, proteins, enzymes, cross linking agents, oligo(ethylene glycol), lipids, nanoparticles, electron density amplifiers, gold clusters, metal clusters, quantum dots, and combinations thereof.
Examples for fluorophores and chromophores that are suitable as probes can be found in Haughland R. P. 1996. Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 6th Ed. (Spence, MTZ, ed.).
Preferably the compounds according to the invention are selected from
wherein (A) is bis-NTA, (B) and (C) is tris-NTA and (D) is tetrakis-NTA (see also FIG. 1), and tautomers, isomers, anhydrides, acids and salts thereof.
In general, said multivalent chelators can be characterized in that several (independent) chelator-groups, nevertheless preferably 2 to 4, such as nitrilotriacetic acid (NTA), can be attached to a molecular scaffold (scaffold G), whereas at the same time a functional group (coupling group X) is provided for coupling to a probe or functional unit F. An important prerequisite for the attachment to probes or functional units is the chemical orthogonality of this coupling group X to the functional groups of the chelator-groups, such as three carboxyl groups in case of NTA. In case of the multivalent chelators as shown in FIG. 1 this was achieved by protective groups at the carboxyl groups (cf. FIG. 2). Now, selective coupling reactions at the additional functional group (X) can be performed without that the chelator-groups are affected.
Furthermore, the object is solved by providing a method for producing the compounds according to the invention. The method comprises the coupling of at least two chelator-groups to the scaffold-structure subsequent or during the synthesis of the scaffold-structure, wherein the chelator-groups can be protected in a suitable manner. During the method according to the invention, the coupling group X can also be suitably protected.
The synthesis of the scaffold-structure according to the invention comprises the synthesis from one or several starting compounds, in particular from amino acids, such as lysine, omithine, 1,3-diaminobutyric acid, 1,2-diaminopropionic acid, glutamate or aspartate and/or their protected derivatives, such as Z-Lys-OtBu, H-Glu(OtBu)-OBzl, Z-Glu-OH.
Further preferred starting compounds are bromoacetic acid-tert-butylester, BOC-□-aminocaproic acid as well as macrocyclic polyamines, such as 1,4,8,11-tetraazacyclotetradecane.
A preferred intermediate is N□,N□-bis[(tert-butyloxycarbonyl)methyl]-L-lysine-tert-butylester (“Lys-NTA-OtBu”, compound 3 in FIG. 25).
The method for production according to the invention preferably first comprises a synthesis of the scaffold-structure. Then, the chelator-groups are coupled to said scaffold-structure. Thereby, the chelator-groups can carry suitable protective groups. Thereby, the scaffold-structure preferably is a cyclic scaffold-structure, such as a cyclam ring structure.
The method for production according to the invention comprises that the scaffold-structure can be composed from protected amino acids or from compounds derived from amino acids. For this, it is preferred that already during the synthesis of the scaffold-structure protected chelator-groups are included. The starting compounds for the scaffold-structure can constitute typical starting products of peptide synthesis that are known to the person of skill. For this, the preferred scaffold-structure is linear or branched (i.e. dendrimeric).
It is preferred that carboxyl-functionalized scaffold-structures with amino-functionalized, protected chelator-groups or amino-functionalized scaffold-structures with carboxyl-functionalized, protected chelator-groups are modified.
In scheme I, a schematic representation of preferred synthesis pathways can be found. In (A), a carboxyl-based scaffold with an amino-functionalized, protected chelator-element (see also FIG. 3A) is modified. The protected functional group (coupling group) X—P is deprotected either selectively (I), or together with the chelator-groups (II), and subsequently can be coupled with a probe or functional unit F. (B) shows an analogous synthesis pathway for an amino-functionalized scaffold that is modified with a carboxyl-functionalized, protected chelator-element (see also FIG. 3B). The intermediates 1 or 2, in turn, can be used as chelator-elements in the first step of the synthesis (see also example 9).

wherein
X is a coupling group,
P is a protective group,
F is a probe or functional unit, and
CL is a chelator-group.
Multivalent chelators with 2, 3 and 4 metal-coordinative-centers were synthesized on the basis of dendrimeric and cyclic scaffolds (i.e. chemical basic scaffolds) (see FIG. 1 and examples).
For the production of bis-NTA-OtBu, tetrakis-NTA-OtBu and a bis-NTA-lipid, preferred are the step of synthesis as depicted in example 9 and FIG. 25.
For the production of Tris-NTA-OtBu and their derivatives with different fluorophores, in particular comprising the fluorophores Oregon Green 488, ATTO 565, FEW-S0387 (for this, see also FIG. 14), the step for synthesis as depicted in example 9 and FIG. 26 are preferred.
Furthermore, the object is solved by the use of the compounds according to the invention for binding to an affinity-tag on a target molecule, whereby the affinity-tag binds to metal-chelator-complexes.
Preferably, the affinity-tag is a peptide-tag comprising between 4 to 15 amino acids. In case of said 4 to 15 amino acids, these are preferably 4 to 15 histidines. Furthermore, 0 to 4 basic amino acids, such as lysine and arginine, can be contained in the peptide-tag.
A preferred affinity-tag is a (His)n-tag, wherein n is an integer between 4 to 15.
Preferred target molecules comprise peptides, polypeptides, proteins as well as peptide and protein-mimetics, and peptide-modified polymers or dendrimers. Regarding this, also a post-translationally modified protein or polypeptide shall be understood by a protein or polypeptide. Peptide- and protein-mimetics comprise compounds that contain similar side chain-functionalities such as peptides or proteins, but are different from those in the composition of the backbone. Possible variations of the backbones comprise a modification of the backbone-atoms (backbone-mimetics), the introduction of bicyclic dipeptide-analogs, and the arranging of the functional groups in a non-oligomeric chain structure (scaffold-mimetics). Oligo-N-alkylglycines (peptoids), for example, belong to the backbone-mimetics, which differ from peptides or proteins in the point of attachment of the side chain (at the N instead of the Cα).
Preferably, the compounds according to the invention can be used for the modification, immobilization, coupling, purification, detection, monitoring, analysis, or for a detection of target molecules in vitro, in vivo, in situ, in fixed and living cells or in lipid vesicles.
A further preferred use of the compounds according to the invention is the controlled and reversible dimerization or oligomerization of target molecules, in particular proteins, to supra-molecular functional units.
The compounds according to the invention can be used in numerous in vitro and in vivo assay methods that are known in the state of the art. Preferred assay methods comprise spectroscopic methods such as absorption spectroscopy, fluorescence spectroscopy, fluorescence-resonance-energy transfer (FRET), fluorescence-correlation-spectroscopy (FCS), fluorescence-bleaching (fluorescence recovery after photobleaching, FRAP), reflectometric interference-spectroscopy (RIfS), surface-plasmon-resonance-spectroscopy (surface plasmon resonance)/BIACORE, optical scanning coupling, quartz-micro balance, surface acoustic waves (SAW), x/y-fluorescence-scanning (FluorImaging) as well as microscopic methods such as fluorescence-microscopy, confocal optical microscopy, total internal reflection-microscopy, contrast enhancing microscopy, electron microscopy, scanning probe microscopy, but also other methods such as magnet resonance spectroscopy, microscopy and tomography, impedance-spectroscopy, field-effect-transistors, enzyme-linked immunoabsorbent assay (ELISA), fluorescence-activated cell or particle-sorting (FACS), radioimmunoassay (RIA), autoradiography, analytical gel filtration, stopped-flow technique, calorimetry, high throughput screening, (HTS), array- and chip-technologies, such as protein-arrays.
Further preferred is the use of the compounds according to the invention for the immobilization of target molecules.
For this, the compounds of the invention can be bound onto a surface or included in a lipid-mono or double layer. Thereby, the surface is preferably selected from glass type-surfaces, such as metalloid oxides, metal oxides and all glass types/glasses, gold, silver, DAPEG-modified glass, PEG-polymer-modified glass or gold, GOPTS-silanized glass, glass type or noble metal surfaces with lipid-mono or double layer, metal selenides, tellurides and sulfides.
By a glass-surface a glass type-surface shall be understood that in addition to glass also comprises quartz, mica, metal oxides, metalloid oxides.
The use of the according to the invention for the production of self-assembled mono layers (SAM) on noble metal surfaces is preferred. SAM are preferably used in methods that are based on surface-plasmon-resonance-spectroscopy, impedance-spectroscopy, scanning probe microscopy, and quartz-micro balance.
Furthermore, the compounds of this invention can be used for the production of functional micro and/or nano-structured surfaces and of protein-arrays.
It is a basic idea of the present invention to use the redundancy of the oligo histidine-tag in order to increase the stability of the protein-chelator-binding by several magnitudes through multivalent chelators (MCH).
The binding of oligo histidine-tags to metal-chelator-complexes in general occurs by a coordinative binding of the N-atoms of the imidazole-residues of the oligo histidine-tags to free coordinative positions in the Ni2+ that is complexed by the chelator, and thus partially coordinatively saturated. In case of Ni-NTA-complexes, four of the overall six coordinative positions of the Ni2+ are saturated by NTA, such that two free coordinative positions for the binding of 2 histidine-residues remain (FIG. 4A). The complex formation thus requires two steps (FIG. 4B): (1) the activation of the chelator by binding of a metal ion, such as e.g. Ni2+, and (2) the binding of histidine-residues of the oligo histidine-tag to the free coordinative positions. By addition of free imidazole in excess the binding of the oligo histidine-tag to the Ni(II)-chelator-complex can be abolished, such that the protein-chelator binding is reversible and switchable. In addition, the chelator can be deactivated by a removal of the Ni2+ from the chelate-complex using EDTA (FIG. 4B).
The binding of the oligo histidine-tag to metal chelate-complexes through only two histidine-residues is relatively unstable (see examples 4 and 8). If metal chelators are immobilized in high density, then several histidine-residues of the oligo histidine-tag can simultaneously bind to several metal-chelate-units resulting to a stronger binding (FIG. 5B). Through the use of multivalent chelators that contain several metal-chelate-units in one molecule, a stable binding to oligo histidine-tags on the molecular level can be achieved. By coupling of further functional units to the chelator, proteins that contain an oligo histidine-tag can be modified stably, but reversibly and switchable (FIG. 5C).
The examinations as described in the following Figures and examples show that the multivalent chelator-compounds represent chemical, highly-affine, switchable molecular recognition structures for affinity-tags, such as the histidine-tag. Thus, they do not only represent an improvement of the traditional chelators, but principally open novel areas of use. Since they can be coupled with nearly any chemical compound, with these chelators nearly any kind of spectroscopic or microscopic probe or functional unit can be attached location-specifically and reversibly an target molecules, such as recombinant proteins. Also, e.g. oligosaccharides, PEG or other biochemical functional units can be reversibly bound to target molecules in order to in this way functionally modify these. In turn, MCH can also be put onto molecular scaffolds and by this way sterically organize target molecules, such as e.g. proteins, in dimeric or multimeric structures. Again, here the versatile switchability is a central feature. Further possibilities result from a coupling with oligonucleotides or PNA. Thus, the multiplexing-possibilities of the DNA-microarrays could be used for the production of protein-arrays.
The following abbreviations are used:
AU absorbance units
Boc tert-butyloxycarbonyl (protective group)
Bzl benzyl (protective group)
DAPEG α,ω-diaminopoly(ethylene glycol)
DIC diisopropylcarbodiimide
DMF dimethylformamide
DIPEA diisopropylethylamine
EDTA ethylenediamine tetraacetic acid
FL fluorescein
FRET fluorescence resonance energy transfer
Glu glutamic acid
GOPTS glycidyloxipropyltriethoxysilane
H10 deca-histidine
H6 hexa-histidine
IDA iminodiacetic acid
Ifnar2 extracellular domain des type I interferon-receptor 2
IFNα2, IFNβ type I interferons α2 and β
MBP maltose binding protein
MCH multivalent Chelator
NTA nitrilotriacetic acid
OG Oregon Green 488®
PEG poly(ethylene glycol)
PNA peptide nucleic acid
RIFS reflectometric interference-spectroscopy
RT room temperature
SAM self-assembling mono layer
TBTU O-(beizotriazole-1-yl)-N,N,N′,N′-tetramethylurollium tetrafluoroborate
tBu tertiary-butyl (protective group)
TEA triethylamine
TFA trifluoro acetic acid
Z benzyloxycarbonyl (protective group)